Rice is one of the most important cereal crops in the world, which is also an important cash crop in China. Nearly 50% of the world's population feed on rice [Khush, 2005]. Rice is mainly distributed in Asia, and has been planted in Europe, America, Africa and Oceania as well. However, rice is also one of the cereal crops that suffer the most from pests. Based on non-exhaustive statistics, the annual loss of rice caused by rice pests such as those from Lepidoptera, etc. has been over 10 million tons globally [Herdt, 1991; Zhu Zhen et al., 1999]. The use of pesticide serves well on pest control, but it also causes severe contamination of toxic residues to human. The pesticide residues in environment may contaminate air and water resources, destruct soil properties; and while killing pests, many beneficial insects are killed as well, which has severely disrupted the ecological balance; moreover, prolonged use of pesticide may also induce resistance in pests; it is therefore believed to be a more environment friendly and more effective control method by means of selectively breeding insect-resistant rice varieties, and enhancing resistance in rice itself. However, due to the lack of insect-resistant resources from rice varieties themselves, and the period of selective breeding of new variety using traditional breeding approach is fairly long, the selective breeding of new insect-resistant rice variety has always been a problem. Recently, with the fast development in cell biology and molecular biology, scientists have successfully integrated exogenous insect-resistant genes into rice genome by transformation using bio-genetic engineering technique, enabling the production of insect-resistant proteins in rice itself that can also be inherited stably in order to control pests [Tu et al., 1998 and 2000; Wei Tang and Zhou Yang et al., 2007]. Such technique can break the species barrier and achieve the direct selection and effective pyramiding of genes, which has substantially improved the breeding efficiency.
Bacillus thuringiensisis is currently the most extensively applied and studied insecticidal microorganism in the world. During the sporulation of Bacillus thuringiensis, it can form and secret some parasporal crystals consisting of insecticidal crystal proteins (ICPs or Cry), which have killing activities specific for arthropods such as insects from Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII) and Diptera (cryIV), etc., as well as animal and plant nematodes (Schnepf et al., 1998; Changyou Li et al., 2007). However, long term growing a unitary Bt gene transgenic crop may also cause resistance in pests. In recent decades, it has been found that different pests show resistance to ICPs and ICPs-transgenic plants at different levels [McGaugher et al., 1985; Van, 1990; Gould, 1992; Lee, 1995; Tabashnik et al., 1997; Wanchao Ni and Sandui Guo, 1998; High et al., 2004; Griffitts J S and Aroian R V, 2005].
More recently, it is found that Bt can secret a new type of insecticidal protein, i.e. Bacillus thuringiensis Vegetative Insecticidal Protein (abbr. VIP) during vegetative growth, which does not form crystal, has no homology with ICPs in the evolution of amino acid sequences, has different insecticidal mechanism from ICPs as well and does not present any similarity in structure either [Estruch et al, 1996; Yu et al, 1997; Estruch et al, 1998; Lee et al, 2003; Estela et al, 2004; Rang et al, 2005]. It exhibits certain insecticidal activity against various agricultural pests from Lepidoptera, Coleoptera etc. [Estruch et al, 1996; Warren et al, 1998], and the insecticidal activity is at nanogram level against certain pests [Rongmei Liu et al., 2004]; additionally, it is also toxic to pests that are insensitive to ICPs such as Agrotis ipsilon [Estruch et al, 1996; Yu et al, 1997]. This provides a new option for controlling agricultural pests that are insensitive or resistant to ICPs.
VIP is a type of extracellular toxic protein that is secreted by Bacillus thuringiensis in its mid-log phase of vegetative growth [Schnepf et al, 1998], and widely exists in Bt in nature. By 2008, 37 species in 8 classes have been identified and isolated [Crickmore, 2008]. Previous studies have shown that these VIPs are relatively conservative genetically. In general conditions, at least 75% of them are present in the supernatant, and compared to ICPs, they are thermally unstable and can be deactivated by treating at 95° C. for 20 min [Estruch et al, 1996].
In the system of nomenclature, according to the homology of their protein sequences, VIPs can be categorized into the following three classes: i.e. VIP1, VIP2 and VIP3 [Crickmore et al, 2008]. VIP1 and VIP2 together construct a binary toxin of insecticidal specificity for insects from Galerucinae in Coleoptera [Warren et al, 1998]; while VIP3 has insecticidal activity against numerous pests from families, genus and species in Lepidoptera on a relatively broad spectrum, with a deep study on its insecticidal mechanism as well [Estruch et al, 1998]. Till 2008, fifty-seven Vip genes have been identified and isolated [Crickmore, 2008].
As a binary toxin, VIP1 and VIP2 function separately in insecticidal mechanism [Barth et al, 2002 and 2004]. Vip2 gene is located at the upstream of Vip1 gene, their products can play their own roles independently [Barth et al, 2004], but only when these two proteins both exist and act synergistically, the maximal insecticidal toxicity of the toxin protein can be achieved [Warren et al, 1998]. Studies have shown that VIP1 can specifically bind to the receptor on mid-gut epithelial cell of insect larvae, and form a channel on cell membrane, providing a route for VIP2 to enter the cytoplasm of the target insect cell [Barth et al, 2004]. VIP2 containing NAD binding sites has ADP-ribosyltransferase activity, which can transfer ribosyl to actin along with the release of nicotinamide, resulting in the blocked polymerization of actin monomers that affect the construction of cytoskeleton, thereby leading to the death of insect cells (Han et al, 1999).
The insecticidal mechanism of VIP3 exhibits primarily on the disruption of the insect mid-gut cells by the toxin [Whalon & Wingerd, 2003]. After being taken up by lepidopteran insects, VIP3A, which is 88 ku in full length, is hydrolyzed and activated by mid-gut trypsin therein. Activated VIP3A protein can bind to unknown receptor molecules (80 ku and 100 ku) on BBMVs of the sensitive larval mid-gut, and form an ion-channel type pore on the mid-gut epithelial cell, which induces apoptosis of insect cell and caryolysis, eventually resulting in the death of insect [Lee et al, 2003]. Moreover, VIP3A protein is soluble once the pH is below 7.5 with its C′ end not being cleaved as well, and these two receptors are also different from any known Cry receptor [Lee et al, 2003]; furthermore, VIP3A may also form a channel on artificial bilayer lipid membranes (BLMs) in the absence of any receptor [Warren et al, 1998; Lee et al, 2003].
Cytopathological experiments have shown that after feeding VIP3A(a) proteins to sensitive insects such as Agrotis ipsilon and Spodoptera frugiperda etc. for 72 h, the mid-gut epithelial goblet cells and columnar cells fall off completely from the basement membrane, resulting in the death of insects [Yu, et al, 1997]. The symptoms caused by Vip3A are similar to those caused by ICPs except a delay in time (Estruch et al, 1996). It thus can be seen that VIP3A has receptors and mode of action that are clearly distinct from ICPs, and the research and utilization of VIP3A are therefore important for expanding insecticidal spectrum, increasing insecticidal toxicity and preventing insects from developing resistance.
The molecular weight of ICP protein is generally 130˜160 ku, and it is only soluble in high alkaline solution of pH>10. It is present in the form of protoxin without toxicity by itself, and after ingestion by insect larvae, it can be dissolved and cut into active polypeptides of 65˜75 ku in the alkaline and reducing environment of mid-gut thereof; in contrast, VIP3A may be cut into active polypeptides of 62 ku by trypsin under weak alkaline conditions, even with pH slightly below 7.5 (Lee et al, 2003). In addition, ICP protein is a crystal protein secreted intracellularly, and VIP protein is an extracellularly secreted protein; during secretion, the N′ end signal peptide of VIP1A protein (100 ku in full length) is cleaved prior to the formation of 80-ku mature toxin, while the N′ end signal peptide of VIP3A protein (88 ku in full length) is usually not cleaved during secretion due to the absence of enzymatic cleavage site [Schnepf et al, 1998]. VIP protein has a broader insecticidal spectrum than Cry protein, and it also shows insecticidal activity against agricultural pests that are insensitive to ICP protein, such as Agrotis ipsilon etc. [Estruch et al, 1996].
Currently, the Vip3 gene that is highly resistant to rice borer has not yet been found and isolated from the natural strain of Bacillus thuringiensis. Accordingly, it is profound to artificially synthesize, modify and create the resource of Vip3 gene that is resistant to rice borer for addressing the insect-resistance duration issue faced by the use of Cry gene [McGaughey, 1985; Van, 1990; Gould, 1992; Lee, 1995; Tabashnik et al, 1997; Wanchao Ni and Sandui Guo, 1998; High et al, 2004].